In vitro inhibitory analysis of consensus siRNAs against NS3 gene of hepatitis C virus 1a genotype

作     者：Imran Shahid Waleed Hassan Al Malki Mohammed Wanees Al Rabia Mohammed Hasan Mukhtar Shaia Saleh R.Almalki Saad Ahmed Alkahtani Sami S.Ashgar Hani S.Faidah Muhammad Hassan Hafeez 

作者机构：Department of Pharmacology and ToxicologyCollege of PharmacyUmm Al Qura University Department of Medical MicrobiologyCollege of MedicineKing Abdul Aziz University Department of BiochemistryFaculty of MedicineUmm Al Qura University Department of Laboratory MedicineFaculty of Applied Medical SciencesAlbaha University College of PharmacyNajran University Department of MicrobiologyCollege of MedicineUmm Al Qura University Department of Gastroenterology and HepatologyFatima Memorial College of Medicine and Dentistry 

出 版 物：《Asian Pacific Journal of Tropical Medicine》 (亚太热带医药杂志（英文版）)

年 卷 期：2017年第10卷第7期

页      面：763-770页

核心收录：

学科分类：1007[医学-药学(可授医学、理学学位)] 100705[医学-微生物与生化药学] 1001[医学-基础医学(可授医学、理学学位)] 100103[医学-病原生物学] 10[医学] 

主　　题：Hepatitis C virus NS3 protein Stable Huh-7 cell culture system RNA interference NS5B HCV therapeutics 

摘      要：Objective: To explore inhibitory effects of genome-specific, chemically synthesized siRNAs(small interference RNA) against NS3 gene of hepatitis C virus(HCV) 1a genotype in stable Huh-7(human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells. Methods: Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin(G418) selection. The cell clones resistant to 1 000 μg antibiotic concentration(G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR. Results: RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested(50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24(80%, P=0.013) and 48 h(75%, P=0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70%(P0.05) viral RNA reduction as compared to NS3-is33, which showed a 64%(P0.05) decrease in viral copy number. siRNA synergism(NS3-is33 + NS3-is44) decreased viral load by 84%(P0.05) as compared to individual inhibition by each siRNA(i.e., 64%–70%(P0.05) in serum-inoculated cells. Synthetic siRNAs mixture(NS5Bis88 + NS3-is33) targeting different region of HCV genom